A confocal microscope uses a point source of light, such as a focused laser beam, to illuminate a point within the sample. Scattered light from the focused spot is then imaged onto a detector through a point aperture such as a pinhole. The light source, illuminated spot and detector aperture lie in optically conjugate focal planes. The size of the detector aperture is matched to the size of the illuminated spot, thereby blocking any light that is not contributed from the back-scatter of the focused spot. By scanning the focused spot over the sample, the detector only receives light from the thin plane at the focus. Light from out-of-focus planes is rejected or spatially filtered by the detector aperture. Optical sectioning is created as long as the sample is optically transparent or translucent. Consequently the confocal microscope can create non-invasive images of thin sections, within turbid, scattering media without having to cut the sample physically into thin slices.
Fiber-bundle based confocal microscopes are capable of imaging biological tissue in near real time, but with a lateral resolution of 2 μm, limited by the fiber spacing, and an axial resolution of 10 μm. K. B. Sung, Fiber optic confocal microscope with miniature objective for in-vivo imaging. Proceedings of the 2002 IEEE Engineering in Medicine and Biology 24th Annual Conference and the 2002 Fall Meeting of the Biomedical Engineering Society (BMES/EMBS), 3:2312-2313, 2002.
An endoscope-compatible confocal laser scanning microscope has been developed that utilizes a gradient index lens system. Gordon Kino, Thomas Wang, Chris Contag, Michael Mandell, and Ning Chan, Performance of dual axes confocal microscope for in vivo molecular and cellular imaging; Progress in Biomedical Optics and Imaging, 5(13):35-46, 2004. J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, and T. Possner, Endoscope-compatible confocal microscope using a gradient index-lens system; Optics Communications, 188(5-6):267-273, February 2001. This system has a reported lateral resolution of 3.1 μm and an axial resolution of 16.6 μm, which is able to resolve some cellular structures in tissues inside the body. However, these resolutions are not suitable for imaging skin.
Line-scanning confocal microscopes have demonstrated excellent imaging capabilities of the human cornea in vivo. C. J. Koester, Scanning mirror microscope with optical sectioning characteristics: Applications in ophthalmology; Appl. Opt., 19:1749-1757, 1980. The line scanner shows promise in imaging skin. However, the line scanner is only confocal in one dimension, thereby decreasing the lateral resolution of the direction of the array.
Raster scanning confocal microscopes for in-vivo imaging are known. See U.S. Pat. Nos. 5,788,639, and 5,880,880. See also M. Rajadhyaksha, R. R. Anderson, and R. H. Webb, Video-rate confocal scanning laser microscope for imaging human tissues in vivo; Appl. Opt., 38:2105-2115, 1999. Such instruments provide good imaging quality with resolutions comparable to that of standard histopathology images (0.5-1.0 μm lateral resolution and 2-5 μm axial resolution). The beam scanning mechanism of these microscopes includes a polygonal mirror, galvanometric mirror, and respective telescopes to place the scan in the focal plane of he objective. Thus, the optics in the beam scanning mechanisms of these instruments tend to be large and bulky and difficult to use.